Biomedical Engineering Reference
In-Depth Information
can be much larger than for TEM. As an example, for small rodent
globes, the whole endothelial (inner) surface of the cornea can be
analyzed by cutting the anterior segment into two pieces [
113
].
After aldehyde fixation, tissues for TEM are postfixed in osmium
tetroxide, which cross-links lipids and thereby stabilizes cellular
membranes. Subsequent processing steps are analogous to those
used for light microscopy, that is, dehydration, embedment (in
epoxy resin), sectioning, and staining. Blocks are sectioned on an
ultramicrotome to produce 80-nm-thick sections that are picked up
on a copper grid and stained using heavy metals such as lead and
uranium [
11
,
105
]. As mentioned before, orientation (semithin)
sections are made for light microscopy first (generally stained with
toluidine blue) to ensure that the area of interest is present and to
further select areas for thin sectioning. Many excellent publications
of standard TEM methods exist [
109
,
111
,
112
,
114
,
115
].
For SEM, aldehyde fixed-tissues are postfixed in osmium
tetroxide and dehydrated through an ethanol series. The tissue is
then dried through critical point drying (using a CPD machine),
mounted in aluminum specimen mounts with the surface of inter-
est facing up and Sputter-coated with a thin layer of conducting
material, typically a metal, such as a gold/palladium (Au/Pd) alloy
[
113
,
116
].
5.3 Processing the
Globe and Ocular
Tissues for Electron
Microscopy
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